Detection and quantification of human herpes viruses

ABSTRACT

In clinical settings as well as in a drug development context, human herpes viruses can be detected, and even quantified, by the use of a real time PCR-based assay. An informatics analysis of existing gene sequences from different HHV types or strains is used to identify a target segment within a gene. A probe oligonucleotide and at least two primer oligonucleotides are then designed for selectively directing the amplification, in the course of a single amplification reaction, of the target segment of a particular HHV type or strain. This method is capable of an unprecedented level of discrimination among the following HHV types and strains: HHV1, drug resistant HHV1, HHV2, drug resistant HHV2, HHV3, HHV4 a , HHV4 b , HHV5, HHV6 a , HHV6 b , HHV7, and HHV8.

BACKGROUND OF THE INVENTION

The present invention relates generally to the detection of human herpesviruses and, more particularly, to measuring human herpes viralinfection in real time, for example, for purposes of clinical testing,drug screening and infection prognostication, inter alia.

Eight human herpes viruses are recognized presently, and these aredivided into three sub-families, as shown below.

1. Alpha herpes virinae Simplex virus human herpes virus 1 (HHV1); humanherpes virus 2 (HHV2) Varicellovirus human herpes virus 3 (HHV3) 2. Betaherpes virinae Cytomegalovirus human herpes virus 5 (HHV5) Roseolovirushuman herpes virus 6 (HHV6); human herpes virus 7 (HHV7) 3. Gamma herpesvirinae Lymphocryptovirus human herpes virus 4 (HHV4) Rhadinovirus humanherpes virus 8 (HHV8)

Various of these viruses are associated with human pathologies. Forinstance, HHV4 or “Epstein-Barr virus” (EBV) is associated withinfectious mononucleosis, also known as “glandular fever.” HHV4 islinked as well with oncogenesis with regard, for example, in Burkitt'slymphoma and nasopharyngeocarcinoma. Additionally, HHV4 is found inimmune-suppressed patients and in patients suffering from Hodgkin'sdisease.

HHV5 is known as “cytomegalovirus” and causes infections in the lungs ofimmune-suppressed persons. HHV5 infection is less common than HHV4, andyet approximately 80% of the population in the United Kingdom, forinstance, experience HHV5 infection by mid-life. HHV5 is probablytransmitted by means of saliva, sexual contact, droplets and bloodtransfusions. In addition, both HHV4 and HHV5 are believed to beassociated with chronic-fatigue syndrome, a malady that may afflict asmany as in six out of every 100,000 people.

HHV6 is associated with “roseola” and “infantum” infections in childrenand with immune-compromised patients. For example, AIDS patients exhibitHHV6 infection, although the significance of the HHV6 infection isunclear. HHV6 is susceptible to antiviral drugs. It is unclear, however,how antiviral drugs work against HHV6 or how resistance to such drugsdevelops. A significant aspect of HHV6 infection is its putative tie-inwith multiple sclerosis (MS) and chronic fatigue syndrome (CFS),respectively.

Less is known about HHV7 and HHV8. No clear evidence for the directinvolvement of HHV7 in any human disease has been reported. Studiesindicate, however, that HHV7 may be associated with HHV6-relatedinfections. In a related vein, HHV8 infection is believed to beassociated with Karposi's Sarcoma.

Conventional techniques have several limitations that have retardedtheir use in elucidating the significance of human herpes virusinfection in the development of several diseases, such as CFS.Prevailing methods are not sensitive for detection of specific types andstrains of human herpes virus and for quantitation of specific humanherpes viruses. If an assay reveals a positive viral load, therefore,additional assays are required to detect specific human herpes viruses.As a consequence, several days may elapse before a patient is diagnosed.In addition, conventional techniques do not detect virus when virallevels are low, for example, at early stages of infection. Accordingly,clinical settings are hampered by a lack of sensitive methods to detectvirus and lose valuable time before a patient can receive treatment.

Current methods also require large amounts of physiological samples,whereas only small amounts of samples from each patient are usuallyavailable for analysis. It is therefore difficult to generatereproducible measurements of viral levels from peripheral blood ofpatients. Additionally, small amounts of physiological samples may bedifficult to obtain and can entail procedures risky to patients, as inthe instance of cerebrospinal fluid, and therefore may not have not beenavailable for routine testing in a clinical environment.

Conventional approaches in this area often involve the polymerase chainreaction (PCR), by which smaller amount of samples can be analyzed.PCR-based testing for human herpes virus has been limited, however, bylong turnaround times. Moreover, the application of PCR to small samplescan lead to an increase in error rates, because more amplificationcycles are required for lesser amounts of sample. In addition, PCR-basedresults for human herpes viruses have appeared to lack reproducibility,given the conflicting date that are reported in the literature.

The absence of a treatment for many human herpes viruses is anotherimportant concern. There is no practical approach to monitoring theeffectiveness of therapeutic agents in the clinical setting, reflectingthe difficulties in testing for specific virus infections and,particularly, in quantitating viral levels. Furthermore, the lack of asuitable screening approach for such therapeutic agents detracts fromthe incentive to develop them.

These difficulties pertaining to conventional PCR-based approaches todiagnosis are illustrated by attempts to measure HHV6 infection. Reportson the use of PCR to measure HHV6 DNA in CFS and MS patients indicatethat the viral levels thus detected depend upon the particularmethodology employed. For example, Locatelli et al., WO 00/29613, usedreal-time quantitative PCR to obtain HHV 6-positive results in 34% ofcerebral spinal fluid (CSF) and 18% of plasma samples from MS patients.Similarly, Soldan et al., Nature Med. 3: 1394-1397 (1997), used a nestedPCR procedure and found that a significant number of plasma samples fromMS patients were HHV6-positive. On the other hand, other labs alsoemploying PCR-based assays reported much lower levels of HHV 6 DNA in MSpatients. For example, Ablashi et al., J. Clinic. Virol. 16(3): 179-191(2000), reported that 9.1% of CSF and 4.5% of plasma samples testedpositive for HHV 6, whereas Taus et al., Acta. Neurol. Scand. 101(4):224-228 (2000), reported that HHV6 DNA was reported absent from CSFdrawn from MS patients.

Similarly, other groups relying on PCR-based methods have reported lowamounts of HHV6 in CFS patients. Secchiero et al., J. Infect. Dis. 171:273-280 (2000), for example, reported that 2.6% of plasma samples fromCFS patients were HHV6-positive.

In summary, prevailing PCR methods for human herpes virus yieldirreproducible results, which complicate efforts to diagnose and treatHHV infection. Further, these methods have not addressed the need for afast throughput assay, which is essential in clinical settings. Asnoted, moreover, there is a lack of therapeutic measures against manyhuman herpes viruses, such as HHV6a and HHV6b, in part due to theabsence of a ready technique for detecting them.

SUMMARY OF THE INVENTION

Accordingly, an urgent need exists for a rapid, sensitive method for theaccurate reproducible detection and quantification of human herpesviruses in patient samples and pooled human plasma. Concomitantly, theability to detect and quantify those viruses would allow assessment ofthe effect of new antiviral agents.

The present invention addresses these needs by utilizing an innovativebioinformatics approach which allows design of sequence-specific primersand probes to measure the presence of a specific viral gene and todistinguish between viral subtypes, such as HH4a and HHV4b or HHV6a andHHV6b. The primers and probes selected through this approach furtherallow discrimination among various HHV strains. Avoiding thedeficiencies of conventional technologies, the present inventionprovides a methodology to detect and to quantify human herpes viruses inreal time (typically, less than one hour), thereby allowing assessment,for example, of treatment options for individual patients in a clinicalsetting. By combining target-gene amplification and target-genedetection in a single reaction, the invention improves the rate andsensitivity for evaluating the presence or absence of specific strainsof human herpes viruses. The improved ability to diagnose HHV offered bythe invention is exemplified by the detection of a particular HHV typein individuals that was undetectable by conventional methodologies.

Target amplification requires the use of forward and reverse primersthat direct Taq Polymerase-catalyzed formation of the complementarystrand of the target. Detection is effected through the use of aninternal fluorogenic probe that hybridizes to the target between the twoprimers. In one embodiment of the invention, the 5′-3′ exonucleaseactivity of Taq Polymerase cleaves a fluorescence quencher moiety fromthe bound probe as it catalyzes the synthesis of the complementarystrand. The cleaved probe dissociates and is detected through anincrease in its fluorescence emission.

The present invention thus provides a method for detecting infection bya particular type or strain of HHV in a sample from an individualsuspected of having HHV, comprising:

-   (a) performing an informatics analysis of existing gene sequences    from different HHV types or strains to identify a target segment    within the gene;-   (b) selecting a probe oligonucleotide and at least two primer    oligonucleotides capable of selectively directing the amplification,    in the course of a single amplification reaction, of the target    segment of the particular HHV type or strain;-   (c) amplifying the target segment in the course of a single    amplification reaction; and-   (d) interpolating the number of HHV viruses of the particular type    or strain in the sample by comparing the number of amplification    cycles required for detection of the target segment to the number of    amplification cycles required to detect a known quantity of the    target segment. In one embodiment, the expression of the gene from    different HHV types or strains is indicative of active HHV    infection.

The method for detecting HHV infection specifically may amplify a targetsegment selected from the group consisting of:

-   (1) a target segment of an HHV1 tk gene comprising SEQ ID NO: 48,    using primers and a probe having sequences set forth in SEQ ID NOS:    1, 2, and 3;-   (2) a target segment of an HHV2 tk gene comprising SEQ ID NO: 49,    using primers and a probe having sequences set forth in SEQ ID NOS:    4, 5, and 6;-   (3) a target segment of a drug resistant HHV2 tk gene comprising SEQ    ID NO: 50, using primers and a probe having sequences set forth in    SEQ ID NOS: 7, 8, and 9;-   (4) a target segment of an HHV3 tk gene comprising SEQ ID NO: 52,    using primers and a probe having sequences set forth in SEQ ID NOS:    13, 14, and 15;-   (5) a target segment of an HHV5 intermediate early gene comprising    SEQ ID NO: 56, using primers and a probe having sequences set forth    in SEQ ID NOS: 25, 26, and 27;-   (6) a target segment of an HHV7 glycoprotein B gene comprising SEQ    ID NO: 60, using primers and a probe having sequences set forth in    SEQ ID NOS: 42, 43, and 44;-   (7) a target segment of an HHV8 K1 gene comprising SEQ ID NO: 61,    using primers and a probe having sequences set forth in SEQ ID NOS:    45, 46, and 47;-   (8) a target segment of an HHV4a EBNA gene comprising SEQ ID NO: 54,    using primers and a probe having sequences set forth in SEQ ID NOS:    19, 20, and 21;-   (9) a target segment of an HHV4b EBNA gene comprising SEQ ID NO: 55,    using primers and a probe having sequences set forth in SEQ ID NOS:    22, 23, and 24;-   (10) a target segment of an HHV6a intermediate early gene comprising    SEQ ID NO: 59, using primers and a probe having sequences set forth    in SEQ ID NOS: 36, 37, and 38;-   (11) a target segment of an HHV6b intermediate early gene comprising    SEQ ID NO: 58, using primers and a probe having sequences set forth    in SEQ ID NOS: 39, 40, and 41;-   (12) a target segment of a drug resistant HHV1 or a drug resistant    HHV2 tk gene comprising SEQ ID NO: 51, using primers and a probe    having sequences set forth in SEQ ID NOS: 10, 11, and 12;-   (13) a target segment of an HHV4 LMP-1 gene comprising SEQ ID NO:    53, using primers and a probe having sequences set forth in SEQ ID    NOS: 16, 17, and 18; and-   (14) a target segment of an HHV6 glycoprotein B gene comprising SEQ    ID NO: 57, using primers and a probe having sequences set forth in    SEQ ID NOS: 33, 34, and 35.

Also provided is a method for cloning a segment of genomic HHV viralDNA, comprising:

-   (a) selecting a candidate HHV gene, wherein expression of the    candidate gene is predictive of active HHV infection;-   (b) performing an informatics analysis of existing gene sequences    from different HHV types or strains to identify a target segment    within the gene;-   (c) selecting a probe oligonucleotide and at least two primer    oligonucleotides capable of selectively directing the amplification,    in the course of a single amplification reaction, of the target    segment of the particular HHV type or strain;-   (d) using said probe oligonucleotide and at least two primer    oligonucleotides to amplify the target segment from isolated genomic    DNA from the particular HHV type or strain; and-   (e) inserting the amplified target segment into a vector.

A polynucleotide molecule of the invention may be selected from thegroup consisting of SEQ ID NO: 1 through SEQ ID NO: 60, and SEQ ID NO:61. Also provided are vectors that comprise a fragment of a gene thatencodes:

-   (1) an HHV1 thymidine kinase protein, wherein the fragment    comprises (i) a polynucleotide molecule having the sequence set    forth in SEQ ID NO: 48, and (ii) up to 30 nucleotide base pairs at    the 3′ or 5′ end of the polynucleotide molecule;-   (2) an HHV2 thymidine kinase protein, wherein the fragment    comprises (i) a polynucleotide molecule having the sequence set    forth in SEQ ID NO: 49, and (ii) up to 30 nucleotide base pairs at    the 3′ or 5′ end of the polynucleotide molecule;-   (3) a thymidine kinase protein from a drug-resistant HHV2, wherein    the fragment comprises (i) a polynucleotide molecule having the    sequence set forth in SEQ ID NO: 50, and (ii) up to 30 nucleotide    base pairs at the 3′ or 5′ end of the polynucleotide molecule;-   (4) a thymidine kinase protein from a drug-resistant HHV1 or a drug    resistant HHV2, wherein the fragment comprises (i) a polynucleotide    molecule having the sequence set forth in SEQ ID NO: 51, and (ii) up    to 30 nucleotide base pairs at the 3′ or 5′ end of the    polynucleotide molecule;-   (5) an HHV3 thymidine kinase protein, wherein the fragment    comprises (i) a polynucleotide molecule having the sequence set    forth in SEQ ID NO: 52, and (ii) up to 30 nucleotide base pairs at    the 3′ or 5′ end of the polynucleotide molecule;-   (6) an HHV4a latent membrane protein-1 or an HHV4b latent membrane    protein-1, wherein the fragment comprises (i) a polynucleotide    molecule having the sequence set forth in SEQ ID NO: 53, and (ii) up    to 30 nucleotide base pairs at the 3′ or 5′ end of the    polynucleotide molecule;-   (7) an HHV4a nuclear protein EBNA2, wherein the fragment    comprises (i) a polynucleotide molecule having the sequence set    forth in SEQ ID NO: 54, and (ii) up to 30 nucleotide base pairs at    the 3′ or 5′ end of the polynucleotide molecule;-   (8) an HHV4b nuclear protein EBNA2, wherein the fragment    comprises (i) a polynucleotide molecule having the sequence set    forth in SEQ ID NO: 55, and (ii) up to 30 nucleotide base pairs at    the 3′ or 5′ end of the polynucleotide molecule;-   (9) an HHV5 intermediate early protein, wherein the fragment    comprises (i) a polynucleotide molecule having the sequence set    forth in SEQ ID NO: 56, and (ii) up to 30 nucleotide base pairs at    the 3′ or 5′ end of the polynucleotide molecule-   (10) an HHV6a glycoprotein B or an HHV6b glycoprotein B, wherein the    fragment comprises (i) a polynucleotide molecule having the sequence    set forth in SEQ ID NO: 57, and (ii) up to 30 nucleotide base pairs    at the 3′ or 5′ end of the polynucleotide molecule;-   (11) an HHV6a intermediate early protein, wherein the fragment    comprises (i) a polynucleotide molecule having the sequence set    forth in SEQ ID NO: 59, and (ii) up to 30 nucleotide base pairs at    the 3′ or 5′ end of the polynucleotide molecule;-   (12) an HHV6b intermediate early protein, wherein the fragment    comprises (i) a polynucleotide molecule having the sequence set    forth in SEQ ID NO: 58, and (ii) up to 30 nucleotide base pairs at    the 3′ or 5′ end of the polynucleotide molecule;-   (13) an HHV7 glycoprotein B, wherein the fragment comprises (i) a    polynucleotide molecule having the sequence set forth in SEQ ID NO:    60, and (ii) up to 30 nucleotide base pairs at the 3′ or 5′ end of    the polynucleotide molecule; or-   (14) an HHV8 K1 glycoprotein, wherein the fragment comprises (i) a    polynucleotide molecule having the sequence set forth in SEQ ID NO:    61, and (ii) up to 30 nucleotide base pairs at the 3′ or 5′ end of    the polynucleotide molecule.

The invention further provides a fluorogenic probe that comprises:

(i) a sequence selected from the group consisting of SEQ ID NOS: 3, 6,9, 12, 15, 18, 21, 24, 27, 30, 31, 32, 35, 38, 41, 44, and 47; (ii) afluorescent reporter moiety covalently attached to the probe; and (iii)a fluorescence quencher moiety covalently attached to the probe.

BRIEF DESCRIPTION OF THE DRAWINGS

Table 1 lists representative primers, probes, and amplicons selected bythe inventive bioinformatics approach. These primers, probes, andamplicons are used for the specific detection of any one of human herpesviruses HHV1, HHV2, acyclovir-resistant HHV2 (DR HHV2), HHV3, HHV4a,HHV4b, HHV5, HHV6a, HHV6b, HHV7 and HHV8.

Table 2 provides primers, probes, and amplicons useful for screeningassays to detect multiple types of HHV. They can be used to detectcombinations of (1) HHV1 and HHV2; (2) HHV4a and HHV4b; or (3) HHV6a andHHV6b.

FIG. 1 illustrates the impact of the bioinformatics approach of theinvention on the choice of primers and probe. FIG. 1A is a phylogenetictree representing genetic relationships among thymidine kinase (tk) genesequences of different HHV1 strains, which are identified by Genbankaccession number. Horizontal lines connecting pairs of sequencesindicate relative sequence homology. (A shorter line indicates greatersequence homology.) Examination of the phylogenetic relationship betweengene sequences and the extent of sequence homology informs the selectionof primers and probes that can be used to detect the gene DNA of thevarious HHV1 strains.

FIG. 1B shows relationships among tk gene sequences from HHV1, HHV2, andHHV3. From these relationships, primers and a probe sequence can bedesigned to detect all HHV1 tk sequences (left cluster). Alternatively,by altering the primer and probe sequences, an assay can be designed todetect both HHV1 and HHV2 sequences (middle cluster), given the relativesequence identity among these sequences. By contrast, HHV3 strains(right cluster) show poor sequence conservation at this target site, andthey are less identical to either HHV1 or HHV2 sequences. Accordingly,the phylogenetic analysis predicts that this particular target would bea poor choice either to detect collectively HHV3 strains or to screenfor the combined presence of HHV1, HHV2 and/or HHV3.

FIG. 2 demonstrates that primers and probes selected by the inventivebioinformatics approach react specifically with the selected targetgene. FIG. 2A depicts real time detection of an LMP-1 gene of HHV4,using an embodiment of the invention that is tailored to the TaqMan®platform. Fluorescence emission intensity (Δ R_(n)) is plotted againstPCR amplification cycle number. The number of PCR cycles required forthe increase in emission intensity is proportional to the amount oftarget sequence in the reaction. C_(T) demarcates the threshold cyclewhere the emission intensity associated with a “positive” detection ofthe target sequence exceeds a preset baseline value (horizontal line).

Positive signals are elicited using HHV4 genomic DNA with primers andprobe complementary to the LMP-1 gene of HHV4 and HHV6 genomic DNA withprimers and probe complementary to the gB gene of HHV6. No positivesignal is detected when HHV6 genomic DNA is used with primers and probecomplementary to the LMP-1 gene of HHV4 or when HHV4 genomic DNA is usedwith primers and probe complementary to the gB gene of HHV6. The primersand probe alone also showed no positive signal.

FIG. 2B depicts assays using the cloned LMP-1 gene amplicon of HHV4 atvarious known concentrations.

Assays of the type depicted in FIG. 2B can be used to create acalibration curve, such as shown in FIG. 2C. The amount of targetsequence present in an unknown sample can then be interpolated bydetermining a C_(T) value for the sample. For example, from the curveshown in FIG. 2C, a sample yielding a C_(T) of 20 cycles is inferred tocontain about 5×10⁴ copies of HHV.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The procedure of the invention allows accurate and sensitive diagnosisof HHV infection in patients. Unlike conventional procedures, infectionby one strain of a specific type of HHV can be distinguished frominfection by another strain of the same HHV type. For example, thedetection procedure of the invention not only can distinguish infectionby HHV6 from infection by HHV7, but it can distinguish infection byHHV6a from infection by HHV6b. Further, the inventive procedure candetect infection by HHV that cannot be detected by conventional PCRapproaches.

An HHV gene is detected within a biological sample by selectiveamplification of a target sequence within the gene, using a set of twooligonucleotide primers and a fluorogenic hybridization probe. Theadvantageous sensitivity and selectivity of the method of the inventionarise from the procedure by which both the target sequence and theprimers and probe sequences are chosen.

The target sequence can be from any HHV gene for the purpose of theinvention; however, the expression of some HHV genes more closelyparallels viral load in an infected individual. Such genes are preferredas the source of the target sequence, because the number of copies ofsuch genes in a biological sample from the infected individual is moreindicative of the level of HHV infection. Genes that fall within thiscategory include but not limited to the thymidine kinase gene (tk gene),the Intermediate Early gene, and some structural genes, such as theglycoprotein B gene. See generally, Weir, Gene 271: 117-30.

Once an HHV gene is selected, a target segment within the gene is chosenfor amplification. Selection of this target segment requires acomparison of available DNA sequences of the target gene from differentHHV types and strains. A growing number of DNA sequences for HHV genesfrom various HHV types and strains are available on public databases,such as GenBank or EMBL. These sequences are aligned using any number ofwell known algorithms, such as the Smith-Waterman algorithm, which isdescribed in Waterman, Bulletin of Mathematical Biology 46:473-500(1984)).

Comparison among HHV sequences is further refined by performing aninformatics analysis of existing gene sequences from different HHV typesor strains to identify a target segment within the gene. Thisinformatics analysis is based on defining phylogenetic relationshipsbetween sequences. When multiple sequences are aligned, gaps arecommonly inserted to optimize the alignment over the length of the totalsequence. The gaps reflect insertion, deletion, or substitution of basepairs in a sequence that arise during evolution. A phylogenetic analysistreats these gaps as “character information,” in the sense thatsequences that are closely related evolutionarily more likely willpossess the same sequence insertions, deletions, or substitutions, whencompared to other sequences. Thus, phylogenetic analysis predicts“clusters” of sequences, such as those depicted in FIGS. 1 and 2, thatpossess common sequence characteristics. For example, in a given targetsegment, a sequence of GATC may be common to all HHV1 sequences, whereasthe corresponding sequence may be CTAG in all HHV2 sequences.Phylogenetic analysis thus classifies sequences by these relatedcharacteristics, allowing identification of sequence segments that maybe useful to distinguish various HHV types and strains.

Phylogenetic algorithms are reviewed in Phillips et al., Mol.Phylogenet. Evol. 16: 317-30 (2000) and Sanderson et al., SystematicZoology, 39: 414-20 (1990). Vector NTI Alignment™ (InforMax, Inc.,Rockville, Md.) is exemplary of the available software programs that canbe used for phylogenetic analysis, pursuant to the present invention.

With respect to the present invention, the phylogenetic analysisidentifies sequences useful for the specific detection of a HHV type orstrain. The distinct clustering of HHV1 sequences and HHV2 sequencesdepicted in FIG. 2, for example, predicts that this particular targetsegment will be useful in distinguishing HHV1 infection from HHV2infection. Knowing the sequences for this stretch of DNA,oligonucleotides can be designed that hybridize to HHV1 sequences, butnot to HHV2 sequences. This design is keyed to the “characterinformation” identified by phylogenetic analysis. Referring to theexample used above, an oligonucleotide that is complementary to GATCwill hybridize to all HHV1 sequences, but it likely will not hybridizeto any HHV2 sequence, because of the four base pair mismatches over thecorresponding four base pair region.

In accord with the invention, each of three oligonucleotides musthybridize to the target sequence to direct its amplification: a forwardprimer, a reverse primer, and a labeled internal probe that hybridizesto the target between the two primers. The specificity of detection isconferred through the hybridization of the internal probe to the targetsequence. It is well known in the art to design a probe that willhybridize to a specified target region, under a given set of conditions.See, for example, Grove, J. Biomolec. Techniques 10(1) (1999). Since theconditions under which PCR reactions are conducted are all more or lessthe same, the base pair composition of the probe is varied so that ithybridizes specifically to the desired sequence. That is, the probe isdesigned to hybridize to the specified target region by varying thecomplementarity between the probe and the target sequence.

The DNA sequence of a useful target segment may not be available foreach HHV strain. However, using the phylogenetic approach of theinvention, the degree of sequence identity of such a strain can beestimated from its phylogenetic relationship with closely related HHVstrains. Thus, the method of the invention can be applied to HHV strainsfor which complete sequence data is unavailable.

HHV infection can be diagnosed by assaying any bodily fluid, such assaliva, serum, plasma, blood, urine, or cerebrospinal fluid. Theinventive procedure also can be used to detect HHV in other sources,e.g. cell culture extracts, tissue samples, plasma fractionationproducts, and pharmaceuticals destined for human or animal use.

Further, the inventive approach can be used to create a screeningplatform to analyze the effectiveness of pharmaceuticals by measuringthe ability of anti-viral agents to mediate human herpes viruspropagation. In situ anti-viral activity analysis is amenable to a rapidthroughput format, which enhances efficiency in identification of testagents that inhibit viral propagation. In addition to determiningspecific activity of anti-viral agents, purification of promisinganti-viral agents can also be tracked. The present invention thuscircumvents problems endemic to ex vivo testing, such as drug toxicityand side effects.

A preferred embodiment of the invention adapts a PCR-based, thermalcycling amplification technology, such as the TaqMan® (AppliedBiosystems) assay, to detect, distinguish, and quantitate human herpesviral DNA. A TaqMan® assay combines PCR and probe-hybridizationreactions into one reaction and is amenable to real-time analysis bymeans of a detection device, such as the ABI 7700, a product of AppliedBiosystems (Foster City, Calif.). See Holland et al., Proc. Nat'l Acad.Sci. U.S.A. 88: 7276-80 (1991), and Livak et al., PCR METHODS ANDAPPLICATIONS 4: 357-362, Cold Spring Harbor Laboratory Press (1995).

Such an assay requires at least three oligonucleotides for the analysisof each target nucleic acid sequence. The sequences of forward andreverse primers are complementary to the ends of the target nucleic acidsequence. A probe sequence is complementary to the sequence foundbetween the ends of the target nucleic acid sequence. A “forward primer”and a “reverse primer” provide a template for polymerase-catalyzedamplification of the target nucleic acid sequence, when hybridized tothe target. A single-stranded oligonucleotide is required for targetdetection and is referred to as the “probe” or “detection probe” or“internal oligonucleotide” or “internal oligonucleotide probe.”

The TaqMan® technique combines two reactions into a single reactionformat: (1) nucleic acid probe hybridization to detect a specific targetnucleic acid sequence and (2) PCR to amplify a target nucleic acidsequence. First, a probe is synthesized so as to contain two distinctmoieties, a fluorogenic reporter group a suitable fluorescence quenchinggroup. Through fluorescence resonance energy transfer, the quencherreduces the fluorescence emission of the fluorescent reporter group. Inthe TaqMan® process, two primers and a probe hybridize to the targettemplate DNA, which then allows polymerase-catalyzed synthesis of thecomplementary strand of the target. In one embodiment, the 5′-3′exonuclease activity of Taq Polymerase cleaves the quencher moiety fromthe bound probe as it catalyzes complementary strand synthesis, causingthe fluorescence emission of the probe to increase, since the reporteris no longer quenched. An increase in the fluorescent signal during theamplification reaction thus depends on the hybridization of both thefluorogenic probe and primers to the target sequence. Targetdiscrimination is enhanced because spurious amplification caused bynon-specific primer hybridization is not detected. See Lee et al.,Nucleic Acids Res. 21: 3761-3766 (1993); TaqMan® PCR reagent Kit,Document No. 430449, published by Applied Biosystems.

The probe is labeled by covalently linking fluorescent tags to eitherend of the probe. A “fluorescent reporter,” such as, FAM(6-carboxy-fluorescein) or TET (6-carboxy 4, 7, 2′,7′-tetrachloro-fluoroscein) is covalently linked to the 5′-end of thedetection probe. A “quencher dye,” such as TAMRA(6-carboxy-N,N,N′,N′-tetramethyl-rhodamine), is attached to thedetection prove. See, for example, Holland et al., Proc. Natl. Acad.Sci. USA 88: 7276-80 (1991); and Perkin-Elmer, AmpliTaq Gold™ ProductInsert, Rev. 3 (1996). An “internal oligonucleotide” or “detectionprobe” or “probe” linked to a “fluorescent reporter” and “quencher” iscalled a “fluorogenic probe” or an “internal fluorogenic probe.”

In accordance with the invention, fluorescent reporters and quenchersother than those mentioned above can be employed in an assay of thepresent invention, see U.S. Pat. No. 5,538,848 to Livak et al. Alsofeasible are other means for generating fluorescent signals, as by theuse of ethidium bromide or another intercalator. See Holland et al.(1991), supra.

Probe sequences and labeling moieties can be adapted to platforms otherthan TaqMan®. A common platform is light cycling, which uses other typesof fluorescent probes well known in the art, including “molecularbeacons,” “smart probes,” “scorpions,” or “eclipse probes.” See, forexample, Thelwell et al., Nuc. Acids Res. 28: 3752-61 (2000). “Smartprobes,” for example, are described in Knemeyer et al., Anal. Chem. 72:3717-24 (2000). These probes report the presence of complementary targetsequences by a strong increase in fluorescence intensity uponhybridization to the complementary sequence on the target. The smartprobes consist of a fluorescent dye, such as oxazine dye JA242, that isquenched by means of complementary guanosine residues at the terminus ofa hairpin oligonucleotide. Specific hybridization to a target sequenceinduces a conformational change in the smart probe forcing thefluorescent dye and the guanosine residues apart, thereby increasing thefluorescence intensity.

Detection devices are available for measuring changes in fluorescenceemission intensity during an amplification according to the presentinvention. Illustrative of such devices are: the ABI 7700 of AppliedBiosystems; the BDProbeTecET fluorescent reader of Becton DickinsonMicrobiology Systems (Sparks, Md.), see Little et al., Clin. Chem. 45:777-784 (1999); and the “SmartCycler” of Cepheid (Sunnyvale, Calif.).This measurement is done in real-time, i.e., in the same time frame thatthe amplificatio reaction. Other methods are available for gaugingchanges in fluorescence which result from probe digestion. Exemplary ofsuch alternative techniques is fluorescence polarization, as described,for example, in U.S. Pat. No. 5,593,867 to Walker et al. Fluorescencepolarization distinguishes larger from smaller molecules based ondifferent rates of molecular tumbling.

By way of illustration, the ABI 7700 “Sequence Detection” softwareemploys three terms to express results: R_(n) (normalized reportersignal), ΔR_(n) (normalized reporter signal minus baseline signal), andC_(T) (threshold cycle). R_(n) represents fluorescence signal fromreporter dye divided by fluorescence signal of a passive reference dye.R_(n) and ΔR_(n) increase as the amount of amplified product accumulatesin the reaction. If initial number of copies of a target nucleic acidsequence in a reaction are high, then fewer number of cycles ofamplification are required to reach a detectable level of fluorescence.

On a graph of R_(n) vs. cycle number, the threshold cycle (C_(T)) occurswhere the application begins to detect an increase in fluorescenceemission signal associated with exponential growth of PCR product.Therefore, C_(T) represents a detection threshold for a sequencedetector. In the ABI 7700 instrument, for example, C_(T) depends onnumber of nucleic acid target sequences at the start of PCR, efficiencyof DNA amplification during PCR, and efficiency of the cleavage of thefluorogenic probe. Although the ABI 7700 device determines C_(T) valuesfrom fluorescence measurements, other signals, as noted previously, canbe used to measure C_(T) values.

For the inventive assay, a calibration curve is prepared using“standards,” which are samples containing a known number of copies of atarget nucleic acid sequence. Independent reactions are performed, eachcontaining a different standards. A graph or a “standard curve” of C_(T)vs. LogN (starting copy number) is prepared using C_(T) values from eachof the reactions that involved a different amount of standard.

The number of copies of target nucleic acid sequence in a test sample isdetermined by interpolating C_(T) values from a reaction containing thetest sample onto the standard curve. In a preferred embodiment, asoftware program, such as the “Sequence Detection” application of theABI 7700, generates a “standard curve” of C_(T) vs. LogN (starting copynumber) for all “standards” and then determines the starting copy numberof unknowns by interpolation. Other software programs can be used in thepresent invention, such as the program within a BDProbeTecET instrument,which reports results through an algorithm as described by Little et al.(1999), supra. The determination of the number of copies of a targetgene sequence in a test sample indicates the number of viruses or viralremnants in the test sample.

In accordance with the present invention, multiplex formats can beemployed to detect more than one target nucleic acid sequence in asingle reaction. For example, primers for more than one target gene,with the corresponding target-specific probes linked to differentfluorescent reporters, can detect multiple targets in a single reaction.

EXAMPLE 1 Designing Primers and Probe for HHV1

This section details an application of the inventive bioinformaticsapproach to HHV1. GenBank lists more than 250 entries of interest forHHV1, including one entry for a complete genome, several open readingframes (ORFs). and plasmid clones. The tk gene of HHV1 was chosen as atarget for assay development, because more than 100 tk gene entries werefound in GenBank. In addition, tk gene activity is known to be a goodmarker of viral infection.

In the region of nucleotides 301-600 of the GenBank sequence entries forHHV1 tk gene, alignment of the sequences revealed few sequencedifferences, indicating that tk genes in different strains of HHV1 shareclose genetic relationships. Phylogenetic analysis (FIG. 1A) confirmedthe likelihood of close genetic relationships within tk genes ofdifferent HHV1 strains.

A segment of the tk gene, referred to as “HHV1 tk gene amplicon” or“HHV1 tk amplicon” (SEQ ID NO 48; see Table 3A), was chosen foramplification, based on TaqMan® assay requirements for nucleotidecontent of primers and probes and for size of amplicon. By the sametoken, TaqMan® assay requirements for nucleotide content andoligonucleotide size guided the design of forward and reverse PCRprimers for amplification of HHV1 tk amplicon, and probe sequence forspecific detection of HHV1 tk amplicon.

Primer and probe sequences and their locations within the HHV1 tk geneare shown in Table 3A.

TABLE 3A HHV1 Thymidine Kinase (TK) Gene Primers and Probes Start LengthTm Sequence HHV1 Forward Primer 358 20 60.10 GTAATGACAAGCGCCCAGAT HHV1Reverse Primer 545 20 59.94 ATGCTGCCCATAAGGTATCG HHV1 Probe 412 20 59.65CGTTCTGGCTCCTCATATCG Amplicon Product Size: 188 (SEQ ID NO: 48) SEQ IDNO: 1 GTAATGACAAGCGCCCAGAT Forward Primer HHV1 SEQ ID NO: 2ATGCTGCCCATAAGGTATCG Reverse Primer HHV1 SEQ ID NO: 3CGTTCTGGCTCCTCATATCG Probe HHV1

A GenBank BLAST search, effected with these primer and probe sequences(SEQ ID NO: 1-3), confirmed that they were unique and complementary tothe HHV1 tk amplicon, SEQ ID NO 48.

The above-described bioinformatics approach, applied to identify the tkgene as a target for HHV1 virus, to identify the tk gene amplicon, andto design appropriate primers and probe for the HHV1 tk gene, likewisewas used in the design of primers and probe for other human herpesviruses. Table 1 presents the results for each of HHV2,acyclovir-resistant HHV2 (DR HHV2), HHV3, HHV4a, HHV4b, HHV5, HHV6a,HHV6b, HHV7 and HHV8.

For some target genes, more than one primer or probe were designed toovercome sequence differences found to exist within different GenBankentries for the amplicon sequence, thereby to allow for detection ofmore strains of a virus.

EXAMPLE 2 Designing Primers and Probes for HHV1 and HHV2 ScreeningAssays

As detailed above, the present invention contemplates a bioinformaticsapproach to the design of primers and probe for screening assays that,pursuant to the invention, will detect multiple types of virus in thecourse of a single reaction. Use of a screen-assay format reduces theinitial number of separate assays that should be required to testdifferent types of virus. Once a screen assay tests positive, thenseparate assays are required to detect individual type of virus thatwere included in the screen-assay design.

This portion of this specification relates the development of ascreening assay, according to the present invention, for detecting humanherpes viruses HHV1, HHV2, and DR HHV2. To this end, the inventors chosethe tk gene as the target; hence, the informatics approach describedabove was performed for the tk gene from the three mentioned viruses,respectively.

GenBank contained more than 100 entries for HHV1 and HHV2 tk genes,including two complete genomes. Incomplete tk gene entries as well asthe portions of the complete genomes from HHV1 and HHV2 were aligned,again as described above. The resultant phylogenetic analysis (FIG. 1B)showed that tk gene sequences from HHV1 and HHV2 are closely related butare diverse from HHV3. Accordingly, HHV3 was excluded from the screenassay design.

Alignment of HHV1 and HHV2 tk genes showed several base pairdifferences, as well as regions containing fully homologous sequences.Therefore, degenerate primers and probe were prepared to overcomedifferences found in the tk gene sequences entries and, thereby, toallow for detection of multiple viral strains. See Table 3B forscreen-assay primer and probe sequences, SEQ ID NOs 10, 11 and 12.

Nucleic acid sequences of screen assay primers and probe were queried,in a BLAST search, against GenBank to confirm that the sequences wereunique and complementary to the intended target, the tk gene.

TABLE 3B HHV1 and HIV2 Screen Assay Primers and Probe Sequences TmLength Sequence HHV1-2 Forward 62.0 20 AGTTGCTGGCCCCCAACGG Primer   CHHV1-2 Reverse 59.4 20 AAACGTGCGCGCCAGGTCGC Primer              G HHV1-2Probe 57.9 20 TTTATCCTGGATTACGACCA 58.1    G             T AmpliconProduct Size: 148 bp (SEQ ID NO: 51) SEQ ID NO. 10 AGYTGCTGGCCCCCAACGGForward Primer HHV1 + 2 screen SEQ ID NO. 11 AAACGTGCGCGCCRGGTCGCReverse Primer HHV1 + 2 screen SEQ ID NO. 12 TTTRTCCTGGATTACGAYCA ProbeHHV1 + 2 screen Nucleotide degeneracy codes: T/C = Y, A/G = R

Essentially the same approach to the design of screen-assay primers andprobes was employed (i) for HHV4a and HHV4b and (ii) for HHV6a andHHV6b. Table 2 shows the primers and probes designed for these HHV4 andHHV6 screen assays.

EXAMPLE 3 Preparing Calibration Curves and Quantifying Number of Copiesof Virus in a Sample

It often will be the case that a polynucleotide can be purchased orsynthsized that corresponds to the segment of the target gene which isselected, via the bioinformatics approach of the present invention.Alternatively, an amplicon selected by the bioinformatics procedure inthe invention can be amplified from an extract of genomic DNA using theamplification primers described above and standard molecular biologytechniques. The amplicon is cloned into a DNA construct, such as,plasmid pCR 2.1, a product of Invitrogen (Carlsbad, Calif.) that has areplication origin, to generate multiple copies of the cloned plasmid.

The cloned plasmid serves as a reference standard for calibration.(“Standards” are samples containing a known number of copies of a targetnucleic acid sequence.) For calibration, inventive assays for concurrentamplification and detection of target gene sequence in the clonedplasmid is performed. Several reactions are performed such thatindividual reactions contain a different concentration of the clonedplasmid. The concentration of plasmid used in each reaction is used tocalculate the number of copies of the target nucleic acid sequence inthe reaction. A graph or a ‘standard curve’ of C_(T) vs. LogN (startingcopy number) is prepared using C_(T) values from each of the reactionscontaining a different amount of cloned plasmid. For a test samplecontaining an unknown number of copies of the target gene sequence, thenumber of copies of the target sequence is determined by interpolatingC_(T) values from a reaction containing the test sample onto thestandard curve. Determination of the number of copies of a target genesequence in a test sample, in this manner, indicates the number ofviruses or viral remnants in the test sample.

EXAMPLE 4 Assay for HHV4

In further illustration of the present invention, an assay is describedfor HHV4. HHV4 belongs to the gamma herpes virinae family and iscommonly known as “Epstein Barr virus” (EBV) or “Lymphocryptovirus.”HHV4 infection usually leads to polyclonal B-cell activation and benignproliferation that maybe sub-clinical or result in infectiousmononucleosis, also referred to as “glandular fever.” HHV4 infection isassociated with oncogenesis, such as Burkitts lymphoma andnasopharyngeal carcinoma. HHV4 infection also is associated with B celllymphomas in immunosuppressed patients, certain T cell lymphomas, andHodgkin's disease.

HHV4 exhibits dual cell tropism: human B-lymphocytes, generally resultsin non-productive infection, epithelial cells, results in productiveinfection. Suitable animal host for experimental purposes areunavailable, instead transformed human cell-lines, HHV4-immortalizedlymphoblastoid cell lines (LCL), are used for HHV4 replication/latencyresearch. HHV4 gene expression in LCL is restricted to about nine of theapproximately 100 genes encoded by HHV4. Genes expressed in LCL arereferred to as “latent genes” because LCL are generally non-permissivefor HHV4 replication. Six of the latent genes encode the nuclearantigens EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C and LP, whereas threeothers encode latent membrane proteins, LMP-1, LMP-2A and LMP-2B.

The process of cellular transformation by EBV is not fully understood.The most abundantly expressed HHV4 latent transcripts in LCL arenon-polyadenylated RNA that do not encode proteins, but are involved inavoidance of interferons. EBNA-1, EBNA-2, EBNA-3A, EBNA-3C and LMP-1 areessential for HHV4-induced B-cell transformation; EBNA-LP and LMP-2Aenhance transformation efficiency. All transforming proteins are notexpressed in Burkitt's lymphoma and nasopharyngeal carcinoma, exceptEBNA-1, which is regularly detected. In addition, several tumors expressLMP-1 and LMP-2.

The above-described bioinformatics approach was applied to HHV4, inorder to allow for the detection of both strain types 1 and 2. Sequencesfrom GenBank were aligned and compared with nearest-neighbor sequences.Based on the above information regarding gene expression in tumors,EBNA-1 and LMP-1 genes were chosen as targets to detect both HHV4strains, and EBNA-1 sequence entries and 45 LMP-1 sequence entries werealigned.

The sequence of primers, probe and amplicon to amplify and detect LMP-1gene of HHV4 in an assay of the invention are shown in Table 2, SEQ IDNOs 16, 17, 18 and 53. Nucleic acid sequences of assay primers and probewere queried, in a BLAST search, against GenBank to confirm that thesequences were unique and complementary to the intended target, theLMP-1 gene.

To confirm that primers and probe selected for the LMP-1 gene of HHV4specifically detected only HHV4 viruses, we also performed assays usingHHV6 genomic DNA and HHV4 primers and probes. Similarly, specificity ofHHV6 primers and probe for HHV6 virus was confirmed using HHV4 genomicDNA in assays with HHV6 gB gene primers and probes. See Table 2, SEQ IDNOS 33, 34, 35, and 57, for primers, probe and amplicon to amplify anddetect gB gene of HHV6. An assay is described below that entailsamplification and detection of the HHV4 LMP-1 gene amplicon in thecourse of a single amplification reaction.

(A.) DNA Extraction

Clones of LMP 1 gene of HHV4 are not commercially available. Therefore,the inventors obtained HHV4 DNA, from strain B95-8, from the Centers forDisease Control (Atlanta, Ga.). Clones of HHV6 DNA are not commerciallyavailable and so viral genomic DNA was extracted from mammalian celllysates infected with virus. Virus-infected mammalian cells wereobtained from The Center for Disease Control.

The method for extracting DNA from 5×10⁷ mammalian cells, using QIAampspin columns (Qiagen catalog No. 51104), is as follows:

-   a. Add 125 μl ProK (20 mg/ml) to 1 ml of sample;-   b. Add 1 ml of Qiagen Buffer AL and vortex;-   c. Incubate at 70° C. for 10 minutes;-   d. Add 1050 μl 100% EtOH and vortex;-   e. Apply 635 μl of sample from step (d) to QIA amp spin column;-   f. Centrifuge the spin column from step (e) at 8000 rpm for 1    minute;-   g. Discard filtrate obtained after centrifugation in step (f);-   h. Repeat steps (e), (f) and (g) as necessary to process the entire    cell lysate;-   i. Place spin column from step (h) in a clean 2 ml collection tube;-   j. Add 500 μl of Qiagen Buffer AW1 to the column;-   k. Centrifuge the column at 8000 rpm for 1 minute;-   l. Place the column in a clean 2 ml collection tube;-   m. Add 500 μl of Buffer AW2 to the column and centrifuge at maximum    speed (5000×g) for 3 minutes;-   n. Place the column in a clean 1.5 ml collection tube;-   o. Elute DNA from the column twice using 200 μl of Qiagen Buffer AE    (preheated to 70° C.) each time;-   p. Incubate eluate at 70° C. for 5 minutes;-   q. Centrifuge eluate at 8000 rpm for 1 minute and discard pellet;-   r. To improve yield of DNA, perform a third elution and repeat    step (o) by reusing the 400 μl solution from step (q); reheat the    solution containing eluted DNA from the third elution at 70° C.,    centrifuge the third eluate at 8000 rpm for 1 minute and discard    pellet.    (B.) Assay for Concurrent Amplification and Detection of Target:

In a preferred embodiment, the TaqMan® assay is used for concurrentamplification and detection of a target gene. The assay uses twoamplification primers and one labeled probe oligonucleotide that areunique and complementary to the target sequence.

The solutions employed in the assay are: 2× TaqMan® Universal Master Mix(Perkin Elmer catalog No. PE 4304437), used at a final concentration of1× in each assay; and, 100 mM dNTP Mix (25 mM each NTP; Perkin Elmercatalogue No. N808-0261), used at a final concentration of 1× in eachassay. For both HHV4 and HHV6, the final concentration of eachamplification primer in the assay was 300 nM and the final concentrationof the fluorogenic probe was 200 nM. The assay was performed in reactionvolume of 50 μl. Sample containing viral genomic DNA was added in amaximum final volume of 5 μl. If lesser than 5 μl of sample containingviral DNA was added to a reaction, then the volume was adjusted byaddition of water. Amplification (polymerase chain reaction) wasperformed by adding 25 μl per reaction AmpliTaq Gold (Applied Biosystemscatalog No. 4304437).

The assay was performed in an ABI 7700 instrument or ABI 7900HTinstrument, programmed as follows: step (1) 50° C. for 2 minutes; 95° C.for 10 minutes; step (2) 40 cycles of amplification of the targetnucleic acid sequence, with denaturation at 95° C. for 15 seconds andannealing/extending at 60° C. for 1 minute, in accordance withmanufacturer's instructions (Applied Biosystems). If a target nucleicacid sequence is present, the fluorescence from the reaction changesduring the course of the polymerase chain reaction. The ABI instrumentsdetect the change in fluorescence in real-time during the amplificationreaction.

In initial experiments, the inventors used HHV4 genomic DNA as templatein the assay described above, with primers and probe corresponding toLMP-1 gene. The primers and probe for the LMP-1 gene, designed pursuantto the above-described bioinformatics approach, are shown in Table 2 asSEQ ID NOs 16, 17, and 18. The red squares and line in FIG. 2A depictassays implemented using HHV4 genomic DNA and LMP-1 gene primers andprobe (C_(T) value was reached in cycle 16).

Primers and probe sequences designed, according to the inventiveapproach, for the gB gene of HHV6 are shown in Table 2 as SEQ ID NOs 33,34, and 35. FIG. 2A also depicts results obtained using HHV6 genomic DNAand primers and probe for the HHV6 gB gene (violet squares and line;C_(T) value is reached in cycle 17). The other symbols in FIG. 2Arepresent controls in which the assay was carried out, for example, withgenomic DNA of HHV6 virus and LMP-1 gene primers and probe.

Thus, FIG. 2A demonstrates that primers and probe designed for the HHV6gB gene, specifically detect HHV6 DNA. FIG. 2A also demonstrates thatHHV6 gB primers and probe recognize different nucleic acid targetsequences on HHV6 DNA and HHV4 DNA, respectively. No amplificationoccurred when viral genomic DNA in the reaction was substituted withwater (C_(T) value is reached in cycle 40).

(C.) Assay Calibration

The foregoing data validate the use of HHV4 genomic DNA as a standardfor assays according to the present invention. To provide a stable andcontinuing source of DNA, for use as a reference standard, a 121base-pair amplicon (SEQ ID NO: 53) for a HHV4 LMP 1 gene segment wascloned between the M13R and M13F primer sequences in plasmid pCR 2.1plasmid, a product of Invitrogen Corporation (San Diego, Calif.). Inthis context, “standard” denotes a sample that contains known copies ofa nucleic acid sequence for amplification in an assay. The number ofcopies of a target sequence in a test sample is determined from astandard calibration curve, and indicates the copy number of viruses orviral remnants in the test sample.

FIG. 2B presents a graph of the inventive assay, implemented usingcloned LMP-1 plasmid DNA and LMP-1 gene primers and probe. Differentconcentrations of cloned DNA were used in the assay. The concentrationof cloned DNA used in individual assays was used to calculate the numberof copies of HHV4 LMP-1 gene targets in each assay. In FIG. 2B, thenumber of copies of HHV4 LMP-1 gene targets used in individual reactionswas varies from 10⁸ to 10. At the highest copy number (10⁸), C_(T) valuewas approximately 15; at the lowest copy number (10), C_(T) value wasapproximately 35.

FIG. 2C presents a graph of a calibration curve for the cloned HHV4LMP-1 gene amplicon. In FIG. 4C, C_(T) values and the correspondingnumber of copies of LMP-1 gene were obtained from the graph in FIG. 2B.In FIG. 2C, the equation that fits the data is: Y=−2.62(X)+36.87, where36.87 is the C_(T) value at infinitely low DNA concentration. Theresults of calibration indicate a linear response in the range of 10copies of amplicon DNA to 10⁸ copies of amplicon DNA, i.e. from 10⁻¹⁵ gto 10⁻⁹ g of DNA.

EXAMPLE 5 Screen Assay for HHV4a and HHV4b

The assay for the LMP-1 gene, described in Example I, detects both HHV4strains that circulate in the human population. Similarly, the inventiveapproach was used to design primers and probe to distinguish the HHV4astrain from HHV4b, also known as “type 1” and “type 2,” respectively.

The genomes of HHV4a and HHV4b are nearly identical. However, thepredicted amino acid sequence of the genes that code for theEpstein-Barr nuclear antigens, EBNA-2, EBNA-3A, EBNA-3B, and EBNA-3Cdiffer between the two types of virus. The predicted primary amino acidsequence of virus type HHV4a differs from that of virus type HHV4b by47%, 16%, 20%, and 28%, respectively. However, most sequence informationfor HHV4, as reflected in GenBank entries, are related to genes that arevirtually identical among the two strains, such as the latent membraneproteins (LMP-1 and LMP-2A), and EBNA-1. Moreover, few of the sequenceentries include information regarding the HHV4 strain used to derive thesequence.

As described above, a region from the LMP-1 gene was developed as thetarget to detect and quantify both HHV4 strains. The inventors likewisechose EBNA2 gene for screen assay development to distinguish betweenHHV4a and HHV4b strains. Of the eight GenBank entries for EBNA2 gene ofHHV4, two contained very short portions (less than 200 bp) correspondingto the EBNA2 gene coding region (see locus HS4NA21 and HS4NA22 in theGenBank database). In addition, only one GenBank entry corresponded toHHV4b (see locus HS4U2IR2).

Sequences encompassing the EBNA2 gene entries were aligned and thegene-coding regions were evaluated for assay development, according tothe present invention. Table 1 presents the primers and probes, thusdesigned using EBNA2, for a screen assay of HHV4. Also see Table 1 forHHV4a and HHV4b amplicons, SEQ ID NOs 54 and 55.

EXAMPLE 6 Analysis of Clinical Samples

An assay of the present invention was performed using DNA extracted fromsamples from HHV4 infected patients. Dr. Joanne Streib, at NationalJewish Hospital and Research Center (Denver, Colo.), kindly provided theHHV4 patient sera. Six sera samples known to be HHV4-positive wereanalyzed, using the primers and probe for LMP-1 gene of HHV4 describedin example 1.

DNA was extracted from the patient sera via the following proceduredescribed below, per CBI protocol DNAREF00015.

-   1. Thaw 1.5 mL of patient serum rapidly at 37° C.;-   2. Transfer 200 μL of thawed serum to a labeled 1.5 mL    microcentrifuge tube;-   3. Add 25 μL of Qiagen protease and 200 μL buffer AL to the serum,    using the Qiagen QIAamp Blood Mini Kit;-   4. Incubate at 70° C. for 10 minutes;-   5. Add 210 μL ethanol (95%v/v);-   6. Centrifuge the above mixture at 6000×g (8000 rpm) for 1 minute,    discard pellet, and apply liquid to a QIAamp spin column in a 2 mL    collection tube;-   7. Discard filtrate and transfer column to a clean 2 ml collection    tube;-   8. Add 500 μL Qiagen Buffer AW1 to the column and centrifuge the    column at 6000×g (8000 rpm) for 1 minute;-   9. Discard filtrate and transfer column to a clean 2 ml collection    tube;-   10. Add 500 μL Qiagen Buffer AW2 to the column and centrifuge the    column at maximum speed 15,000×g for 3 minutes;-   11. Discard filtrate and place column in a clean, labeled 1.5 mL    collection tube;-   12. Add 200 μL Qiagen Buffer AE (preheated to 70° C.) to elute DNA    from the column; incubate column at room temperature for 1 minute,    and then centrifuge at 6000×g (8000 rpm) for 2 minutes;-   13. Use the eluted DNA immediately, or store at −20° C.

Assays were performed to amplify and detect LMP-1 gene of HHV4 induplicate aliquots of each DNA extract; see Table 4 for results. Toconfirm that the extracted DNA was a suitable template for purposes ofthe present invention, each extract also was assayed for beta-actingene. (Applied Biosystems.)

TABLE 4 Assay results for HHV4 DNA extracted from patient samplesTaqMan ® Copies of LMP-1 Patient C_(T) Value DNA per mL 1 33.1   27,40035      5,200 2 31.3   133,600 30.9   189,900 3 36.4    1,510 37.2     0 4 31.5   112,000 32.1   67,000 5 40        0 34.6    7,300 6 28.71,433,000 28.6 1,313,000

FIG. 2 illustrates that an assay of the present invention, employing theprimers and probe designed for HHV4, has the sensitivity needed tomeasure as few as 10 copies of target DNA. The results also show thatthe primers and probe designed by the inventive approach detect thepresence of HHV4 viral DNA in patient sera.

The level of viral target DNA detected in patient serum is expected tovary depending on the stage of the disease. High levels of viral DNAwould characterize situations where patients are actively sheddingvirus, while patients with a chronic infection or an infection inremission would have low levels of viral DNA. From the data of Table 4,therefore, the inventors concluded that patient 6 was in a moreinfectious stage than patients 5, 1, and 3. The source of the patientsera, Dr Streib, confirmed this analysis, based on her assessment ofclinical symptoms.

EXAMPLE 7 Second Analysis of Clinical Samples

Pursuant to the present invention, sera from patients diagnosed with MSwere assayed for HHV4, HHV6, and HHV7, using primer and probe sets shownin Table 1. Dr. Jacquelyn Friedman, at the Rockefeller University (NewYork City, N.Y.), kindly provided the patient sera. The inventors wereinformed that the samples had tested negative for HHV6 by a nestedPCR-based technique.

DNA from the patient samples was extracted according to the protocoldescribed above. Assay results for duplicates of each sample are shownin Table 5.

TABLE 5 Results of assay of HHV4, HHV6 and HHV7 in DNA extracted fromsera of patients diagnosed with MS HHV4 Mean HHV6 Mean HHV7 Copies/mLCopies/mL Copies/mL MM <10   6580  <350   RP <10  66,300  <350   VY <10160,000  <350   JS <10  33,100  <350   ES <10 393,000  <350   KO <10 99,400 1350 LR <10  16,800 2000 AZ not assayed 515,000 1750 JN notassayed   8990 1700 AK not assayed  12,000 1750 RU not assayed   79302300 WM not assayed   4420 1700 EN not assayed 127,000 2150 EL notassayed  37,600 1850

The assay results in Table 5 reveal that no patient tested positive forHHV4 (detection limit was 10 copies/mL). Some patients (9/14) werepositive for HHV7 (detection limit was 350 copies/mL), and all patientstested positive for HHV6 (detection limit was 10 copies/mL).Concentration of HHV6 in the samples ranged from 4000 to 500,000copies/mL.

IgM assays had previously demonstrated that 18/43 (42%) of the patientsamples tested positive for IgM to HHV6 virus, and that seventeen of thesamples had been taken in the course of a viral attack or during theprogress of infection. Five of seventeen samples from those activepatients were positive (30%). Therefore, IgM assays detected activestages of viral infection only 30% of the time (in five samples), ascompared to 17 samples detected by the inventive procedure.

EXAMPLE 8 Third Analysis of Clinical Samples

Dr. Dharam Ablahsi of Advanced Biotechnology, Inc. (Rockville, Md.)kindly provided cell lysates of mammalian cultured cells infected withHHV4, HHV5, HHV6a, HHV6b, or HHV7 virus. Dr. Ablashi also providedmiscellaneous patient samples, cord blood, serum, and cerebrospinalfluid for assay.

DNA was extracted from each sample, according to the protocol describedabove. Assays of the invention were performed for HHV4, HHV5, HHV6,HHV6a, HHV6b and HHV7 viruses, using appropriate primers and probe sets(see Table 1).

The assay results, set out in Table 6, show that cells infected withHHV6a strain GS, for example, tested positive for HHV6 and HHV6a, buttested negative for HHV6b. This conforms to expectations, because theprimers and probe for the HHV6 screening assay are designed to detectboth HHV6a and HHV6b strain variants. Also as expected, HHV GS testedpositive in the HHV6a assay. Conversely, all cells infected with HHVZ-29 (an HHV6b variant) tested positive in the HHV6 screen assay and inthe HHV6b assay, but tested negative in the HHV6a assay. Thus, theinventive approach was effective in distinguishing between these strainvariants.

TABLE 6 Results of assay using cultured cells or viral samples HHV4 HHV5HHV6 HHV6a HHV6b HHV7 Sample (DNA copies per mL) Uninfected 0 0 0 0 0 0cells (HSb2; sup't) HSb2/HHV6a 0 0 1.80e9 9.1e8  0 0 Sup't/HHV7 0 0 0 00 1.3e9 HHV6a (GS) 0 0 2.75e6 6.49e7 0 0 HHV6b (Z-29) 0 0 5.2e7  0 3.7e70 HHV4 (P3HR) 1.79e9 3,667 2,667 0 0 0 HHV5 20,333 4.01e8 0 0 0 0 (AD169)

Table 7 shows that bodily fluids from patients diagnosed with chronicfatigue syndrome (CFS) tested positive for HHV6 and HHV7 but not forHHV4 or HHV5. The diagnostic utility of the inventive assays isreflected in the fact that the results shown in Table 7 are consistentwith results from other studies which seek to establish the associationof CFS with HHV6, and the co-incidence of HHV7 infection. See, forexample, Ablashi et al., J. Clin. Virol. 16: 179-191 (2000). The utilityof the assay is also demonstrated by the ability to make such adiagnostic prediction using samples from CSF, peripheral blood monocytecells (PBMC), plasma, and serum.

TABLE 7 Results of assay using patient samples HHV4 HHV5 HHV6 HHV6B HHV7Diagnosis Fluid (DNA copies per mL) CFS Plasma 0 0 40,067 0 31,167 CFSPlasma 0 0 45,033 0 70,000 GFS Plasma 0 0 38,067 0 61,867 CFS Plasma 0 049,967 0 82,300 CFS Plasma 0 18,700 64,533 0 79,400 Unkn. CSF 0 0 2.08e60 0 Unkn. CSF 0 0 110,676 0 0 Karposi's Serum 0 0 47,800 0 56,600sarcoma Unkn. PBMC 0 0 1.68e6 0 0 Unkn. PBMC 0 0 83,333 0 0

Table 8 illustrates that the assay of the invention is advantageouscompared with methodologies previously employed in the art. Regularreverse transcriptase PCR failed to detect HHV6 in a biological samplefrom four individuals contained HHV6. The procedure of the inventionunambiguously detected HHV6 in three of the four individuals.Accordingly, the assay of the invention improves the ability to detectHHV beyond the capabilities of current technology.

TABLE 8 Assay results, expressed in copies/mL of HHV6, for conventionalreverse transcriptase PCR reaction and assay of the invention. PatientConventional RT PCR assay Assay of the invention 1 Negative 44,000 2Negative 42,450 3 Negative 23,370 4 Negative    0

TABLE 1 Primers, probes, and amplicons for assays of individual HHVtypes SEQ ID NO NAME LENGTH SEQUENCE 1 HHV1 TK Forward Primer 20GTAATGACAAGCGCCCAGAT 2 HHV1 TK Reverse Primer 20 ATGCTGCCCATAAGGTATCG 3HHV1 TK Probe 20 CGTTCTGGCTCCTCATATCG 48 HHV1 TK Amplicon 188 4 HHV2 TKForward primer 19 CTCCGAGACCCTGACGAAC 5 HHV2 TK Reverse primer 20GGCGTGCTGATTGTTATCTG 6 HHV2 TK Probe 19 ACACGCAGCACCGTCTGGA 49 HHV2 TKAmplicon 114 7 DR HHV2 TK Forward primer 20 ATCAGCGTCAGAGCGTTCCC 8 DRHHV2 TK Reverse primer 21 GGACGTAGACGATATTGTCGT 9 DR HHV2 TK Probe 24GTAGAAGCGGATATGGCTTCTCGC 50 DR HHV2 TK Amplicon 327 13 HHV3 TK Forwardprimer 20 GTATTGGCGTAACCTTGCAG 14 HHV3 TK Reverse Primer 20CATAATTGCATGCGGAGAAC 15 HHV3 TK Probe 20 AGACGCACAACGCCTCACGG 52 HHV3 TKAmplicon 148 25 HHV5 IE Forward primer 24 TGCAGAGCATGTATGAGAACTACA 26HHV5 IE Reverse primer 20 CAGCCATTGGTGGTCTTAGG 27 HHV5 IE Probe 21GAAGCCATCCACATCTCCCGC 56 HHV5 IE Amplicon 235 42 HHV7 glyB Forwardprimer 21 GCTGACTTTGTCATGACTGGA 43 HHV7 glyB Reverse primer 18AGACGCGCAAGAAACCTC 44 HHV7 glyB Probe 21 TGTTCAATTGCCAGCGGGACA 60 HHV7Amplicon 108 45 HHV8 K1 Forward primer 19 TCGTSTCGCCTGTCAAATC 46 HHV8 K1Reverse primer 23 ATCCTTGGTACACACCMTAG 47 HHV8 K1 Probe 27TTCTTGTATTTATGACRCTCGTAGCTC 61 HHV8 Amplicon 226 19 HHV4a EBNA Forwardprimer 20 GTCCAGTCCTCGGTCTTCAT 20 HHV4a EBNA Reverse primer 20GAGCCTCTGGGCTATTATGG 21 HHV4a EBNA Probe 20 CTCCTGGCCCATCGAATGCC 54HHV4a EBNA Ampicon 22 HHV4b EBNA Forward primer 20 CCCGTCTGTAGAGTGACACC23 HHV4b EBNA Reverse primer 20 GCCCTCCCAACTTTCATCTA 24 HHV4b EBNA Probe20 CCCAGTGATTGGTATCCTCCAACGT 55 HHV4b EBNA Amplicon 116 36 HHV6a Forwardprimer 22 GTTGAAGGGACAGAACAAGATG 37 HHV6a Reverse primer 20GCAGCTGAATCAGAGTTTGC 38 HHV6a Probe 34 CGGCACCCTATGAGAGTGAAAGCG 59 HHV6aAmplicon 152 39 HHV6b Forward primer 21 AACTCCAAGTGTACCGAAACG 40 HHV6bReverse primer 21 GGTGCTGAGTGATCAGTTTCA 41 HHV6b Probe 26TGTGATGGTTTCCATGACAACCCTTT 58 HHV6b Amplicon 221 In SEQ ID NOS: 7, 8, 9,and 50, DR = “drug resistant”. Nucleotide degeneracy codes for HHV8 are:C/G = 5; A/C = M; A/G = R

TABLE 2 Probes for screening assays of the invention SEQ ID NO NameLength Sequence 10 DR HHV(1+2) Forward primer 20 AGTTGCTGGCCCCCAACGG   C11 DR HHV(1 + 2) Reverse primer 20 AAACGTGCGCGCCAGGTCGC              G12 DR HHV(1 + 2) Probe 20 TTTATCCTGGATTACGACCA    G              T 51 DRHHV(1 + 2) Amplicon 205 16 HHV(4a + 4b) LMP-1 Forward 19GCACCCTCAACAAGCTACC primer 17 HHV(4a + 4b) LMP-1 Reverse 21TAGGTTTTGAGAGCAGAGTGG primer 18 HHV(4a + 4b) LMP-1 Probe 24CTAACTCCAACGAGGGCAGACACC 53 HHV(4a + 4b) LMP-1 Amplicon 121 28 HHV(6a+ 6b) IE Forward primer 23 GAGAGTGAAGATGAAGAGGATGG 29 HHV(6a + 6b) IEReverse primer 22 TTATTGGGATGGTAAACACTGG 30 HHV(6a + 6b) IE Probe 1 25TCATCTGACTCGCTGCTCGATTCAG 31 HHV(6a + 6b) IE Probe 2 25TCATCTGACTCGCTACTCGATTCAG 32 HHV(6a + 6b) IE Probe 3 25TCATTTGACTCGCTGCTCGATTCAG 33 HHV(6a + 6b) gB Forward primer 20GGGAATTTGGCAGAATCTTG 34 HHV(6a + 6b) gB Reverse primer 20GACCGTAAACCTGAGACACG 35 HHV(6a + 6b) gB Probe 27GGAACGATAACGATGTTGCACGAACTT 57 HHV(6a + 6b) gB Amplicon 106

1. A set of polynucleotide molecules wherein the set comprises thepolynucleotide molecules consisting of SEQ ID NOS: 33, 34, and 35 andoptionally a fourth polynucleotide molecule comprising SEQ ID NO:
 57. 2.A set of polynucleotide molecules according to claim 1, furthercomprising polynucleotides consisting of SEQ ID NOS: 1, 2 and 3, andoptionally a polynucleotide molecule comprising SEQ ID NO:
 48. 3. A setof polynucleotide molecules according to claim 1, wherein the set isfurther comprising polynucleotides consisting of SEQ ID NOS: 4, 5 and 6,and optionally a polynucleotide molecule comprising SEQ ID NO:
 49. 4. Aset of polynucleotide molecules according to claim 1, further comprisingpolynucleotides consisting of SEQ ID NOS: 7, 8, and 9, and optionally apolynucleotide molecule comprising SEQ ID NO:
 50. 5. A set ofpolynucleotide molecules according to claim 1, further comprisingpolynucleotides consisting of SEQ ID NOS: 10, 11 and 12, and optionallya polynucleotide molecule comprising SEQ ID NO:
 51. 6. A set ofpolynucleotide molecules according to claim 1, further comprisingpolynucleotides consisting of SEQ ID NOS: 13, 14, and 15, and optionallya polynucleotide molecule comprising SEQ ID NO:
 52. 7. A set ofpolynucleotide molecules according to claim 1, further comprisingpolynucleotides consisting of SEQ ID NOS: 16, 17, and 18, and optionallya polynucleotide molecule comprising SEQ ID NO:
 53. 8. A set ofpolynucleotide molecules according to claim 1, further comprisingpolynucleotides consisting of SEQ ID NOS: 19, 20, and 21, and optionallya polynucleotide molecule comprising SEQ ID NO:
 54. 9. A set ofpolynucleotide molecules according to claim 1, further comprisingpolynucleotides consisting of SEQ ID NOS: 22, 23, and 24, and optionallya polynucleotide molecule comprising SEQ ID NO:
 55. 10. A set ofpolynucleotide molecules according to claim 1, further comprisingpolynucleotides consisting of SEQ ID NOS: 25, 26, and 27, and optionallya polynucleotide molecule comprising SEQ ID NO:
 56. 11. A set ofpolynucleotide molecules according to claim 1, further comprisingpolynucleotides consisting of SEQ ID NOS: 36, 37, and 38, and optionallya polynucleotide molecule comprising SEQ ID NO:
 59. 12. A set ofpolynucleotide molecules according to claim 1, further comprisingpolynucleotides consisting of SEQ ID NOS: 39, 40, and 41, and optionallya polynucleotide molecule comprising SEQ ID NO:
 58. 13. A set ofpolynucleotide molecules according to claim 1, further comprisingpolynucleotides consisting of SEQ ID NOS: 42, 43, and 44, and optionallya polynucleotide molecule comprising SEQ ID NO:
 60. 14. A set ofpolynucleotide molecules according to claim 1, further comprisingpolynucleotides consisting of SEQ ID NOS: 45, 46, and 47, and optionallya polynucleotide molecule comprising SEQ ID NO:
 61. 15. The set ofpolynucleotide molecules according to claim 1, further comprisingpolynucleotide molecules consisting of SEQ ID NOS: 16, 17, 18, 25, 26,27, 42, 43, and 44, and optionally polynucleotide molecules comprisingSEQ ID NOS: 43, 56 and
 60. 16. A method for detecting infection by HHV6in a sample from an individual suspected of being infected with HHV6,comprising: (a) amplifying, in the course of a single amplificationreaction, a target segment of an HHV6 glycoprotein B gene comprising SEQID NO: 57 using primers and a probe consisting of SEQ ID NOS: 33, 34,and 35 and (b) interpolating the number of HHV6 viral nucleic acidcopies in the sample by comparing the number of amplification cyclesrequired for detection of the target segment to the number ofamplification cycles required to detect a known quantity of the targetsegment.
 17. A method for detecting infection by either HHV6a or HHV6bin a sample from an individual suspected of being infected with eitherHHV6a or HHV6b, comprising: (a) amplifying, in the course of a singleamplification reaction, a target segment of an HHV6 glycoprotein B genecomprising SEQ ID NO: 57, using primers and a probe consisting of SEQ IDNOS: 33, 34, and 35; and (b) interpolating the number of either HHV6a orHHV6b viral nucleic acid copies in the sample by comparing the number ofamplification cycles required for detection of the target segment to thenumber of amplification cycles required to detect a known quantity ofthe target segment.